ABSTRACT Largemulti-centerGWAstudieshavefoundassociationsbetweenover20genomiclociandlate-onset Alzheimer?sdisease(LOAD).However,theprecisetargetgenes,thecausalgeneticvariantsandtheir molecularmechanismsofactionthroughwhichtheyexerttheirpathogeniceffectsremainlargelyunknown. Ourlong-termgoalistoelucidatecausalgeneticfactorsandtheirfunctionaleffectsthatcontributetotheriskof developingLOAD.Ourcentralhypothesisisthatchangesinexpressionlevelsofcriticaldiseasegenesisan importantmolecularmechanismunderlyingLOADetiologyandthatcausalvariantsmodulateexpressionof thesediseasegenes,andbythatcontributetoLOADrisk.ChangesingeneexpressioninLOADvs.healthy controlsweredescribedinbraintissuesbyourteamandothersandpreviousstudiesreportedthecis- associationsoftaggingSNPswithexpressionofnearbyLOAD-riskgenes,providingastrongscientificpremise fortheproposedstudy.Inthisstudy,wewillemployamultifacetedapproachthatcombinesinsilico,invitro andinvivomethodstoinvestigateregionsinthegenomethatweresignificantlyassociatedwithLOAD-riskin GWAstudies.InAim1wewillidentifytargetgeneswithinLOAD-associatedregionsthatshowdifferential expressionalongtheneuropathologicalprogressionofLOAD.Wewilldeterminetheexpressionprofileof geneswithintheseregionsinneurons,astrocytesandmicrogliaisolatedfromaffectedandunaffectedrapidly autopsiedhumanbraintissuesusinglasercapturemicrodissection(LCM)coupledwithnCountersinglecell geneexpressiontechnology(NanoString).Aim2willdiscoverregulatorynoncodingsequenceswithinLOAD- associatedregions.First,wewillprioritizecandidateregulatoryelementsusingbioinformaticstoolsandhuman genomedatabases,aswellasATAC-sequencingexperimentsusingNeuN+/-nucleifromaffectedand unaffectedhumanbraintissuestodeterminechromatinaccessibilityprofilesincelltype-andpathological stage-specificmanners.Thefunctionalityofthecandidatenoncodingsequenceswillbethencharacterized usingiPSC-derivedmodelsystemsthatwillbegenomeeditedtocarrydeletionsofthepredictedregulatory sequences.Aim3willfocusonShortStructuralVariants(SSVs)andwillinvestigatethefunctionaleffectsand causalityofSSVsinthecandidateregulatorysequences.WewilluseSMRTsequencingcombinedwithCas9 system(PacBio)toaccuratelydeterminetheSSVsgenotypeandhaplotypesinLOADcomparedtocontrol subjects,andwillexaminetheirregulatoryeffectsusinggenomeeditedisogeniciPSC-derivedneuronsand/or astrocytesmodelsthatcarrydifferentalleles/haplotypesattheSSVsite.Ourstudywilladvancethe identificationofcausalgeneticfactorsandtheunderstandingoftheirmoleculareffectsthatcontributetothe riskofdevelopingLOAD.Thisknowledgewillprovideinsightregardingactionabletargetsfordevelopmentof noveltherapiesforLOAD.Furthermore,theidentifiedSSVswilladvancethedevelopmentofgenetic biomarkersforearlydiagnosisandforenrichmentofclinicaltrialswithsubjectsathighrisk.